Cxcr4-sgRNA/Cas9 RNP-transfected WHIM HSPCs have a selective advantage for durable hematopoietic reconstitution over mock-transfected cells in lethally irradiated Cxcr4^+/w recipient mice. (A) Competitive gene therapy experimental design. The editing status and CD45 isoforms used for distinguishing donor and recipient cells are given at the tops and bottoms of the mouse icons, respectively. Left-Cxcr4-sgRNA/Cas9 RNP-transfected (edited) and mock-transfected (mock) HSPCs were mixed 1:1 and transplanted into lethally irradiated Cxcr4^+/w recipients. (B) Cxcr4-sgRNA/Cas9 RNP–transfected (edited) and mock-transfected (mock) donor HSPCs were transplanted in approximately equal numbers. Numbers indicate the percentage of total cells in each gate before transplantation. (C) Hematopoietic reconstitution of the blood. The leukocyte subsets are indicated above each panel. Each data point represents an individual mouse and the solid lines trace the means across time for the experimental conditions coded at the right of the panel. (D-E) Hematopoietic reconstitution of the BM. Mature BM cells (D) and HSPCs (E) were analyzed on day 381 after transplantation when the experiment was arbitrarily terminated. In panel D, data for the same leukocyte subsets in the blood are regraphed from the same day from panel C to facilitate comparison. Data are presented as a scatter plot (n = 3) and as the mean ± SEM of the percent of total donor-derived cells for each subset. Data are from a single experiment, representative of 3 independent experiments. LT-HSC, long-term hematopoietic stem cell (CD34^-Flt3⁻LSK); MPP, multipotential progenitor (CD34⁺Flt3⁺LSK); ST-HSC, short-term hematopoietic stem cell (CD34⁺Flt3⁻LSK).